Method to simultaneously detect different antibodies and antigens in clinical alimentary and environmental samples

ABSTRACT

Method to simultaneously detect different antibodies and antigens via immunoenzimatic tests and ELISA (Enzyme Linked ImmunoSorbent Assay) constituted by small absorbent cylinders, on which the immunocomplexes are formed, blocked at a modular distance on a probe; that carries a label to identify the sample under examination.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending application Ser. No.10/711,847, entitled: “Device and Method to Simultaneously DetectDifferent Antibodies and Antigens in Clinical Alimentary andEnvironmental Samples,” filed on Oct. 8, 2004, the contents of which areincorporated herein by reference, which is a continuation ofInternational Application No. PCT/IT03/00218, entitled: “Device andMethod to Simultaneously Detect Different Antibodies and Antigens inClinical Alimentary and Environmental Samples,” filed on Apr. 9, 2003,which International Application claims the benefit of priority to priorfiled Czech Republic patent application Serial No. 2002A000002 (CZ)filed on Apr. 11, 2002.

SUMMARY OF THE INVENTION

This invention refers to a method to simultaneously detect differentantibodies and antigens. Said method is based upon the use of a devicewhich allows the introduction of the solid phase of an immunoenzimaticreaction directly into the sample to be examined, thus inverting theprocedure of the first step in the execution of the immunoenzimaticmethods and in those of ELISA (Enzyme Linked ImmunoSorbent Assay), whichgenerally foresee that the sample be distributed among the microwells ofmicroplates, on the surface of which the single type (solid phase),reagent, antigen or antibody is adsorbed.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a series of eight adsorbent.

FIG. 2 shows the rod with attached cylinders immersed into the samplecontainer containing a test sample with antigens and antibodies.

FIG. 3 shows a test rod, after incubation in a test sample, placed inthe notches of the grill for handling and/or transport.

FIG. 4 depicts the grill being placed on a microplate.

FIG. 5 depicts the grill placed on a microplate.

FIG. 6 shows the grill lifted from the microplate for cleaning.

FIG. 7 shows a rod with four small cylinders and a label. The rod islaid on the twelve columns of the microplate from one side to the other,doubling the microplates capacity to carry out analyses on double thenumber of samples. The small colored squares are symmetrically repeated.

FIG. 8 depicts the testing process.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

In the proposed method the solid phase carries diverse adsorbed reagents(antigens and antibodies) for various simultaneous analyses and isrepresented by the surfaces of small protruding cylinders from a rod,which is introduced directly into the sample and later, afterincubation, placed on a screen for microplates or microstrips, which ismoved from the microwells of a first microplate, which contains theconjugate, to a microplate (or microstrip) where the chomogenous sublayer is distributed. This last, once removed from the screen that holdsthe small cylinders, then passes through a spectrophotometer for areading: this allows the use of equipment that is already on sale and isnormally used in laboratories for analysis and dose not constitute,therefore, an economic obstacle to the wider distribution of theinnovation.

The small cylinders can be single and assembled on the probes, accordingto the analytical necessities, or those of research, or the can be setout in numbers of 4 or 8 or 12 for each rod, according to specificpanels that respond to the principal diagnostic exigencies (FIG. 1).

The versatility of the proposed invention makes the method of varioustypes of approach, both experimental and routine, extremely flexible andadaptable, and permits the slimming down and optimization of laboratoryfields that foresee the use of immunoenzimatic tests and ELISA.

State of the Art

The methods and the device used up to the present date for the detectingof antibodies and antigens, that foresee the use of immunoenzimatictests and ELISA, present some very delicate steps that condition theanalytic result and which need particular care. There are:

Cleaning the solid phase, constituted of microwells of small dimension;

Drying the same solid phase;

The different times of contact of the reagents that are manuallydistributed in the microwells, with the consequent possibility ofsystematic errors.

Furthermore, these tests are conditioned by:

Processing times for the samples that suffer in the time necessary forthe distribution of the various samples among the microplates and forthe transcription of the identifying codes;

The volume of samples on which to execute the analysis, which limits thesensitivity of the test, and which cannot be varied because it dependson the number of the microwells of the microplates;

The necessity to execute the various analyses for a single sample indifferent microplates.

To overcome these inconveniences, several solutions have been proposed,among them those described in the following patent documents: DE4120139, EP-A1-0301141, EP-A-0087899.

In DE 4120139 microwells are made on a microplate, arranged in diverseparallel lines and columns. A secondary cover for the microplate createsa structural support that binds the antigen or antibody, also disposedalong lines and columns corresponding to the geometry of the microwells,so that by covering the microplate with the supporting cover theypenetrate perfectly into the microwells. In each dimple of theindividual microplates a sample to be analyzed is placed by pipette. Thecover that holds the supporting structure for the antigen again coversthe microplate, and it is allowed to incubate for sufficient time toallow the formation of the immunocomplex. Once formed, the immunocomplexremains stuck to the distal part (the beads) of the support. The firstcleaning of the supports occurs inside the microwells. The entire cover,together with the supporting structures for the antigen, is transferredonto another microplate, in which the conjugate antispecies has beendistributed, and it is left to incubate once again. If the immunocomplexis present, it will bind to it. A second cleaning is performed. Thecover is once again transferred onto a third microplate, in which thechromogenous sub layer has been distributed. Finally, it is put aside toincubate to permit the chromogenous action to happen.

The samples to be analysed must be quickly deposited singularly onto thefirst microplate.

In EP-A1-0301141, a variety of specific antibodies for differentantigens can be simultaneously determined in a single clinical sample.

The invention comprises:

A support structure that is preferably plastic, provided with a group ofopenings ellipsoidal in shape;

A porous membrane of immobilised nitrocellulose on said support, onwhich the different antigens are sprayed;

Binders (double adhesive layers) to immobilize said membrane on thesupporting member;

A first container, containing the diluted sample of serum/whey to beanalysed;

A second container containing the conjugated antispecies;

A third container that contains the chromogenous sub layer.

The sustaining member, on the membrane of which the different antigensare adsorbed, is inserted into the first container that holds the sampleto be analyzed. It is allowed to incubate for five minutes, during whichtime the formation of the immunocomplex that remains adhered to themembrane takes place. There follows a phase of cleaning the supportmember with distilled water, to eliminate the non bound residues, andlater the support is inserted into a second container in which theconjugate alkalin-phosphatase antispecies has been distributed. Therethen follows an ulterior incubation, during which the conjugate binds tothe immunocomplex (said aggregate, a conjugate-immunocomplex, willalways remain adhered to the support). The third cleaning then takesplace. In the final phase the supporting structure is inserted into athird container that contains the chomogenous substrate. The alkalinephosphatase (an enzyme of the conjugate) converts the solublechromogenous sub layer into an insoluble colored product that is deposedonto the porous membrane. The products so bound are visible in the formof colored marks on the porous membrane. The absence of the coloredmarkings on the membrane indicates the absence of specific antibodies inthe clinical sample.

In EP-A-008799, microwells disposed in a number of parallel columns andlines are laid out on a microplate. A secondary cover over themicroplate presents supporting structures that constitute the solidphase for anchoring the immunocomplex that are removable, thoughconnected by a breakable appendix these too, are laid along lines andcolumns that correspond to the geometry of the microwells, in such a waythat when the microplate is covered with the cover then the supportstructures penetrate perfectly into the microwells. In each dimple thesample to be analyzed is distributed. The microplate is covered againwith the cover that sustains the support structures for the antigen andit is allowed to incubate for sufficient time to permit the formation ofthe immunocomplex. Then follow the phases of the ELISA methodology.

Some of the methods analyzed do no resolve the problem of thedistribution of the samples among microplates and the transcription ofthe identification codes, with a notable loss of time and with thepossibility of error, while others do not consent the execution of theanalyses for the search for antigens in the samples.

Another disadvantage is represented by the fact that the incubation timestarts only from the moment when the microwells in the microplates havebeen filled.

To overcome these inconveniences and disadvantages, and to optimize theanalytical procedure, the present device and method for simultaneousdetecting of different antibodies and antigens is proposed, which, byway of example but not limited to, is used for:

-   -   the simultaneous search for different antibodies in:

Mass samples of milk;

Individual blood samples (animal or human) with an anticoagulant;

Samples of saliva

Egg samples;

-   -   the simultaneous search for different antigens (micro-organisms        of interest in the medical or veterinary fields or their toxins,        xenobiotic substances etc.) in:

Pathology samples;

Food samples;

Environmental samples

-   -   the simultaneous search for different antibodies and different        antigens in:

Mass samples of milk;

Individual samples of blood (animal or human) with an anticoagulant;

Saliva samples;

Egg samples;

Pathology samples

The Sensitization of the Solid Phase

The solid phase is constituted by the surfaces of the small cylinders ofplastic material that are suitable for use in immunoenzimatics(polystyrene, nylon, acrilonitric styrne etc.) end is sensitized usingthe current methods which foresee the contact of the surface to besensitized with the protein reagent (antibody or antigen) in a tampon ofcarbonatelbicarbonate of pH=9:5 and incubation for a night atrefrigerator temperature.

The substantial difference between this sensitizing procedure in respectto the classic method consists in the fact that the containers utilizedfor sensitizing the small cylinders are filled with different reagents(whilst in the classic method all the microwells of a microplate aresensitized with the same reagent) in such a way that each small cylinderfrom the same probe brings diverse adsorbed reagents to the surface. Forexample, if one uses a microplate with 96 small microwells for thesensitization of the small cylinders carried by 12 rods, each with 8small cylinders, then the microplate would be charged in the followingmanner:

Line A: reagent A in all 12 microwells

Line B: reagent B in all 12 microwells

Line C: reagent C in all 12 microwells

Line D: reagent D in all 12 microwells

Line E: reagent E in all 12 microwells

Line F: reagent F in all 12 microwells

Line G: reagent G in all 12 microwells

Line H: reagent H in all 12 microwells

In this way, at the end of the procedure for the sensitizing of thesolid phase, each of the 12 rods with 8 small cylinders is useful forcarrying out 7 different tests plus a negative control contemporarily,and each rod will have an identical sequence.

The solid phase prepared in this way is conserved in a refrigeratoruntil the moment of analysis.

The Analysis Samples

A series of polymer small cylinders 2, made of polystyrene or nylon, areattached to rod 1, made of plastic or metal, by clipping joint 3 on thecylinder to notch 4 on rod 1, as seen in FIG. 1. While eight smallcylinders 2 are depicted, it is envisioned that four or twelve couldalso be used. The rod 1 and cylinder 2 assembly is inserted into samplecontainer 5 a, being immersed into a test sample contained therein, asshown in FIG. 2. The cylinders are sensitized, prior to placement in thesample with a different protein reagent (antibody or antigen), for thesample for the search for antigens and antibodies. To identify thesamples, label 6 can be detached from rod 1 and inserted into lid 5 b ofsample container 5 a. During the transport time for the sample'scontainer 5 with the rod 1 furnished with small cylinders 2 is used asincubation time for the formation of the immunocomplex on eachindividual small cylinder 2.

Upon completion of the incubation period, rod 1 and small cylinder 2assembly is removed from sample container 5, and label 6 is returned torod 1. Rod 1 with the small cylinders 2 is placed on grill 10, formed byat least three parallel horizontal sides (11, 12, and 13) and with atleast two parallel vertical sides (14 and 15) and with a handle (16) forlifting and/or transport, as seen in FIG. 3. For the housing of therods, twelve notches area available, equally distanced on the horizontalsides 11, 12, and 13, and eight notches equally distanced on thevertical side 14 and vertical side 15. Colored button 16 is displayed ongrill 10 to indicate the direction of loading the rods onto a on it. Ongrill 10, rods 1 are laid out according to colored button 16.

Rods 1 are placed in the notches present on parallel horizontal sides11, 12, and 13 of grill 10, seen in FIG. 4. Grill 10 is placed onmicroplate 20 is shown, and placed in microwells 21. The lines are alsodistinguished by differently colored small squares 22.

Grill 1 is of a shape and size sufficient to consent to the loading ofrods 1 in such a way as to make the position of small cylinder 2coincide with microwells 21, disposed on microplate 20, as illustratedin FIG. 5. After use, grill 10 is lifted from microplate 20 forcleaning, seen in FIG. 6.

To increase testing capacity of microplate 20, rod 1 a, with four smallcylinders 2 and with label 6 is placed on microplate 20. The same rod 1a is flipped and placed on the twelve columns of the microplate 20, fromone side to the other and doubling the microplates capacity to carry outanalyses on double the number of samples. Consequently colored squares22 are symmetrically repeated are modified on the microplate.

The overall process is shown in FIG. 8. Rod 1 a, with small cylinders 2,are immersed in a test sample, contained in sample container 5, seen inFIG. 8(1). The cylinders are incubated in the sample, seen in FIG. 8(2),then washed three times in wash containers 7, shown in FIG. 8(3). Rod 1a is then placed on microplate 25 a, such that small cylinders 2 alignand fit into microwells 21, seen in FIG. 8(4). Color squares 22 areaffixed to the side of microplate 25 a, allowing identification of testsamples. The cylinders 2 are incubated in the microwells, depicted inFIG. 8(5). The cylinders are washed again in wash containers 7, shown inFIG. 8(6). Rod 1 a is returned to microplate 25 a, and incubated, seenin FIGS. 8(7) and 8(8). Rod 1 a is removed from the microwells, depictedin FIG. 8(9).

The sample (for example blood with an anticoagulant, or milk) iscollected in bottles or test-tubes (in which is placed an equal quantityof diluting liquid) into which rod 1 is immerged—in a specificpredisposed lodging on the inside of the lid cover that then closes thesample's container—bringing the small cylinder where the antigens areadsorbed towards the sample to be examined for antibodies (and/or toascertain the presence of antibodies directed towards antigens): eachsmall cylinder holds a different antigen (or a different monoclonalantibody) and can be distinguished by a particular coloring (FIG. 2);one small cylinder is not sensitized with any antigen and acts as anegative control (the probes can be assembled in such a way as tocontain—an antigen plys the negative control;—up to 7 antigens plus thenegative control, as shown in the illustration;—up to 11 antigens plusthe negative control, inverting the direction of the microplates andusing rods with 12 small cylinders).

The cover or lid for the samples' containers is also furnished, on theoutside with a specific holder for the card bearing the sample'sidentification code; said card is placed on the cover at the moment thesample is taken and, at the time of the sample's processing, is takenfrom the cover and put onto the rod. The time involved in the laboratoryfor transporting the samples is used as a period of incubation for theformation of immunocomplex, if there should be specific antibodiespresent in the sample when they confront the adsorbed antigens in thesmall cylinders (and/or the specific antigens for the monoclonalantibodies adsorbed in the small cylinders).

Should the sample prove positive for one or more antigens (orantibodies) present on the rod that has been inserted, the immunocomplexthat forms adheres to the surface of the respective small cylinder bythe specific antigen (or antibody); in fact, the reaction makes use ofthe adsorbment of the antigent (or antibodies) at the solid phase,represented by the small cylinders. This solid phase that detains theimmunocomplex goes through further steps. On arrival at the laboratory,the container holding the sample is opened, the rod bearing the smallcylinder is removed, and provided with its own card bearing theidentification code for the sample, then placed, together with the otherrods from different samples, on a specific holder in the form of a grillfor microplates (FIG. 3); in the case of a single sample it will beplaced on a support for microstrips.

A thorough cleaning takes place, bathing the small cylinders with aspecial solution and the cleaning liquid is left to drip dry, placingthe extremity of the small cylinder onto blotting paper (it issufficient to place it onto the paper). In this manner any traces ofmatter adhering to the surface of the small cylinders is removed, sinceit is not specifically bound to them, while the antibodies (or theantigens) working in the formation of the immunocomplex remain adheredto the specific antigens (or antibodies) adsorbed by the small cylinderssurface.

The conjugate anti-species is dispensed among the microwells of amicroplate or microstrip that has not adsorbed any kind of reagent, andis immediately covered by the holder bearing the rods, in such a waythat the small cylinders dip into the reagent and the color of the smallcylinders corresponds to a colored strip born on the side of themicroplate or the microstrip holder (FIG. 4, FIG. 5), should one want touse holders marked by color (in the case of the search for the antigensin the sample under examination, the conjugate will be constituted of anenzyme bound to a monoclonal or polyclonal antibody directed at anepitope of the antigen that is different to the one that binds to themonoclonal antibody adsorbed onto the small cylinders' surfaces). It isleft to incubate (generally 30′ at +37° C.).

During the incubation period the conjugate, that is the antibody boundto an enzymatic protein capable of catalyzing the chromogenic reactionin the presence of the specific sub layer, binds specifically to theimmunocomplex whenever this has been formed during the precedingincubatory phase with the rod inserted in the sample to be examined.Should the sample be positive then the conjugate will also remainadhered to the surface of the small cylinders and will be transportedwith them in the final microplate.

After incubation the holder bearing the rods is taken away and a furthercleaning and drying takes place, to eliminate the reagents that do notadhere specifically to the small cylinders' surfaces, while theantigen/antibody/conjugate complex (or that of theantibody/antigen/conjugate complex) remains firmly adhered.

The holder bearing the rods is placed onto another, final, microplate ormicrostrip, where the chromogenous sub layer has been dispensed.

It is left to incubate (generally 10-15 minutes at room temperature), toallow catalysed reaction of the enzyme bound to the conjugate thatbrings to the development of a colored substance (chromogenous reaction)to take place, the quantity of which is proportional to the quantity ofthe immunocomplex that adheres to the small cylinders and the opticaldensity of which is measurable by spectrophotometer. The chromogenousreaction is simply blocked by lifting the supports; that is, byextracting the small cylinders that hold the conjugate adsorbed with theenzyme (FIG. 6).

A spectrophotometer reading of the microplate or microstrip is taken.The same procedure is followed for the probes with 4 or 6 or 12 smallcylinders (FIG. 7).

Should one want to carry out the method exclusively in a laboratory,omitting to insert the rod bearing the small cylinders directly into thesample and collection container to be analyzed, one can proceed to thedistribution of the samples into the microwells (into which an opportunequantity of diluting liquid can eventually be placed) of a microplatethat has not been sensitized by any reagent, by using a micropipette. Inthis case as many replicates of the sample must be distributed as thereare small cylinders for the rods that will successively be inserted intothe microwells containing the sample, and the same direction of placingthe rods inserted into the frill must be observed (for example, in thecase of 8 small cylinder rod, in a microplate 12 samples aredistributed, each one replicated 8 times, that is in the 8 microwellsthat form a column). Once the distribution of the samples among themicroplates has taken place, the grill containing the rods bearing thesmall cylinders is placed, in such a way that they dip into the samplesto be analyzed and it is left to incubate at a suitable temperature forthe necessary time. At the end of the incubatory period one proceeds tothe cleaning of the small cylinders, and immerges them into themicrowells of the microplate that contain the specific conjugates andthey are allowed to incubate at a suitable temperature for the necessarytime. One then proceeds to a further phase of cleaning the smallcylinders and their immersion into the microwells of the microplate thatcontain the chromogenous sub layer; they are left to incubate for thenecessary time at a suitable temperature, the grill containing theprobes with the small cylinders is lifted, and one proceeds to take aspectrophotometer reading.

Summarizing the procedure, as shown in FIG. 8, a series of adsorbentcylinders 2 attached to rod 1 a are inserted into a sample tube 5 with acollected sample. After incubation, the cylinders are washed, as shownin FIG. 8(3) and probed as shown in FIGS. 8(4) to 8(9).

Advantages and Merits of the Methods of this Invention

The execution time for this type of test in a laboratory is about 50minutes in total, while the classic method takes decidedly longer,thanks to the necessity of transferring a proportional amount of thesample onto the microplate, transcribe the identification codes for thesamples in the order of their distribution on the microplate, allowingthe samples to incubate with the adsorbed antigen at the solid phaseconstituted by the walls of the microwells of the microplate, everythingto be multiplied by a number of times equal to the number of tests to becarried out for the same sample.

Two cleanings occur, as with the classic method.

The incubatory periods, following the addition of the conjugate andafter the addition of the chromogenous sub layer, ware equal to those ofthe classic method.

An extra microplate is necessary with respect to the classic method,non-sensitized by any type of reagent and therefore of very low cost.

The cost of the rods bearing the small cylinders onto which the antigens(our antibodies) are adsorbed is comparable to that of the microplatessensitized with antigens or antibodies.

The cost of the holders for the rods bearing the small cylinders shouldonly be considered an initial cost, such as a cost for laboratorymaterial (just as for test-tubes racks).

Merits of the Method

The advantages offered by the proposed invention are distinguished ingeneral advantages—that is, in the improvements to the quality of theclassic immunoenzimatic method—and in advantages of a specific kind;that is, relative to specific applications in the field of health.

General Advantages

The optimization of the cleaning procedures during the solid phasederives from the possibility of investing the small cylinders with aflow of cleaning solution far more efficient in the removal ofnon-specific reagents capable of altering the reaction;

The optimization of the drying procedures during the solid phase andconsequently the conformity of the small cylinders, that allows theliquid in which they had been previously immersed to drip easily;

The reduction of systematic errors deriving from the contact of eachsample of the microplate with the reagents at the same time;

The reduction in the processing times for the sample is made possible byusing the transport time to the laboratory as a first-step period ofincubation;

The reduction in the processing times for the sample is, furthermore,tied to the abolition of one step in the procedure, given that the solidphase, to which the conjugate is eventually anchored, is extracted fromthe final microplate, making the inoculation of the solution that blocksthe chromogenous enzymatic reaction unnecessary;

An improvement to the test's sensitivity derives from the possibility ofincreasing the quantity of the sample to be analyzed according to will,without increasing the quantities of reagents necessary to carry out thetest; the quantity of the sample under exam is separated, therefore,from the quantity of the reaction, and this brings about an increase inthe sensitivity of the test, above all in mass milk samples, in foodsamples, and in environmental samples (in the case of food andenvironmental samples, the possibility of predisposing monoclonalantibodies capable of reacting with the un-denatured bacterial antigensshould be studied; this would make it possible to use the proposedmethod in a greater quantity of samples compared to that used in theclassic immunoenzimatic method, thus avoiding the need to fall back onthe enriching phase—the release of the analytical report 24 hours priorto that of the classical method—or to eliminate the phases ofpre-enrichment and enrichment, with the release of the analytical report48 hours ahead of the classical method; it remains to be carefullyevaluated, however, the significance to be attributed to the positivereactions that are not preceded by a phase of growth of bacteria).

Specific Advantages:

The possibility of processing battery samples, that is at the same timefor the entire range of tests to be carried out (breathing panel,enteric panel, panel of animal health during weaning, pathogens andtoxins in foodstuffs etc.) responds in a congruous manner to the needsof diagnostic serology and of food and environmental control, thatrarely foresee on single type of test per sample, allowing the releaseof the complete analytical report at the end of one single test.

The solid phase, constituted by the small cylinders, can be planned toallow the assembly of the individual small cylinders at any time,according to the diagnostic needs that occur on each occasion.

Precautions

In the case of carrying out mixed tests in the search for differentantigens in the same sample, together with a search for differentantibodies or not, it should be noted that while the same conjugateanti-species can be used in the search for any type of antibody, in thesearch for the antigens a specific conjugate is used for each type ofantigen; in the first microplate used in the laboratory on the arrivalof the sample, a specific conjugate is placed on each line or column toshow up specific antigens; to make this distribution easier, the bottomof the microwells of the microplate can be colored, give that thismicroplate does not go through a spectrophotometer reading.

Other characteristics and advantages of the invention will appear clearfrom the following description of several methods for constructing theinvention, given only an non-limiting examples in the FIGS. 1, 2, 3, 4,5, 6, 7 and 8.

As illustrated in FIG. 8, the method lends itself to the creation ofkits for the examination of samples in the field or in laboratories(medical or veterinary), thanks to the simplification of the proceduresfor the distribution of the sample, the cleaning at the solid phase, andto the possibility to carry out simultaneously the detection of moreantibodies and/or antigens. In this case the spectrophotometer readingcan be substituted by a visible reading of the results of the test.

FIG. 8 represents the steps to carry out for an analysis of a single rod(1 a) with four small cylinders (2).

Step 1. Taking the sample and introducing the rod into the container (5)(eventually graduated and already containing the dilution solution).

Step 2. Incubation at room temperature.

Step 3. Three passages in test-tubes (7) containing the cleaning liquid.

Step 4. The introduction into a microstrip (25 a) that already containsthe specific conjugates.

Step 5. Incubation at room temperature.

Step 6. Three passages in test-tubes (7) containing the cleaning liquid.

Step 7. The introduction into a microstrip (25 b) that already containsthe chromogenous sub layer.

Step 8. Incubation at room temperature.

Step 9. The visible reading of the results.

On each microplate (25 a) or (25 b) made from a single column ofmicrowells, that is a microstrip, the antibodies or antigens to besought are indicated by specific small colored squares (22)—for example,antigen A, antigen B, monoclonal antibody, no reagent (negativecontrol)—the color of which corresponds to that of the small cylinders(2).

The Preparation of Positive Controls

For each microplate or series of microplates or microstrips, thepositive control is prepared by previously placing a rod inincubation—the small cylinders of this rod are immersed in microwellsthat contain the specific reagents for the positive control of the test(antigens and antibodies)—for the necessary period and at a suitabletemperature (the small cylinder that has not been sensitized by anyreagent is inserted into a dimple without any specific reagent andserves as an ulterior negative control); the rods on which theimmunocomplex has formed can eventually be conserved, ready for use. Thepositive control rod is then inserted into the grill in which the rodsextracted from the samples are loaded, and is examined at the same timeas these rods, and with them follows the above described testprocedures. The invention, it must be noted, is not limited in use tothe examples give in the illustrations, but can be modified andperfected by anyone skilled in the art without breaking patent.

1. Method to simultaneously detect different antibodies and antigen viaimmunoenzimatic test and ELISA—Enzyme Linked ImmunoSorbent Assay,wherein a solid phase on which an immunocomplex forms is constituted byadsorbent cylinders disposed on a rod; said adsorbent cylinders areplaced on the rod and sensitized with different reagent, immerging theminto microwells of a microplate; said adsorbent cylinders of each rodonce sensitized, said rod is inserted into a container with a sample tobe analyzed; that once the incubation period for the sample in thecontainer has terminated, each rod is placed on a support; saidadsorbent cylinders of each rod on the support are cleaned; saidadsorbent cylinders of each rod on the support are inserted into themicrowells of a microplate, containing specific conjugates at a suitabletemperature for a period of incubation; that, once the incubationterminates, the adsorbent cylinders on the support are lifted from themicroplate and cleaned; said adsorbent cylinders from each rod on thesupport are inserted into the microwells of a microplate, containing achromogenous sub layer at a suitable temperature for a period ofincubation; that the small cylinders of each rod on the support areextracted and the results are read.
 2. Method to simultaneously detectdifferent antibodies and antigens via the immunoenzimatic tests andELISA ELISA—Enzyme Linked ImmunoSorbent Assay, according to claim 1,wherein a container for the sample to be analyzed is constituted by amicroplate that has not been sensitized and that on such a microplatethe grill, furnished with rods bearing the small cylinders is placed.For a suitable incubation period at the necessary temperature.